title: [Preparation of monoclonal antibodies against flagellin core protein of Vibrio vulnificus and its application in testing Vibrio vulnificus from food products].
abstract: OBJECTIVE
  To prepare the monoclonal antibodies (mAbs) against flagellin core protein of Vibrio (V.) vulnificus and establish the double-antibody sandwich ELISA for testing V. vulnificus from food products.
  
  
  METHODS
  BALB/c mice were immunized by flagellin which was extracted by differential centrifugation method from V. vulnificus ATCC 1.1758. The splenocytes taken from the immunized mice were fused with Sp2/0 myeloma cells when the antibody titer reached 1:32 000 in serum. The hybridoma cell lines were prepared and screened by hybridoma technique and ELISA. The cells secreting mAbs were cloned through the limited dilution. The hybridoma cell lines were used to generate ascites. The mAbs were obtained by purification from the ascites.
  
  
  RESULTS
  Five hybridoma cell lines which stably secreted mAbs against flagellin were isolated and named VVNo.1-VVNo.5. The mAb titer in serum reached 1:(2×10(6);). SDS-PAGE showed that the relative molecular mass (Mr;) of flagellin protein was 44 000, and that the purity was high. Double-antibody sandwich ELISA was set up using VVNo.5 antibody, and the sensitivity reached 10(3); CFU/mL culture broth. The ELISA showed that VVNo.5 antibody was highly specific to V. vulnificus. The detection limit was 2 CFU/25 g culture broth in artificial contaminated samples.
  
  
  CONCLUSIONS
  The mAbs were obtained against flagellin core protein of V. vulnificus. The double-antibody sandwich ELISA was established using VVNo.5 mAb. The monoclonal antibody of VVNo.5 was highly specific to V. vulnificus, without cross reaction with non-target bacteria. Therefore the monoclonal antibodies of VVNo.5 could be widely used in detecting V. vulnificus from food samples as well as the clinical samples.
authors.affiliation: Beijing Entry-Exit Inspection and Quarantine Bureau, Beijing 100026, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zeng
authors.forename: Jinxia
authors.initials: J
authors.lastname: Cheng
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Lin
authors.initials: L
authors.lastname: Zhang
authors.forename: Haiyu
authors.initials: H
authors.lastname: Zhang
authors.forename: Haiyan
authors.initials: H
authors.lastname: Wei
authors.forename: Xuesong
authors.initials: X
authors.lastname: Liu
authors.forename: Dong
authors.initials: D
authors.lastname: Cao
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005408:Flagellin / Q000276:immunology*; D005516:Food Microbiology; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D041261:Vibrio vulnificus / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D000911:Antibodies, Monoclonal; D005408:Flagellin